SUPPLEMENTARY VIDEO LEGENDS
Hyaluronan activates Smad4/Hyal-2/WWOX signaling and causes bubbling cell death when the signaling complex is overexpressed, Hsu et al.
Video 1. Smad4/WWOX/p53 signaling. DU145 cells were transiently transfected with ECFP-Smad4, EGFP-WWOX and DsRed-monomer-p53 expression constructs. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration).
Video 2. Smad4/WWOX/p53 signaling – merged images of FRETc and bright field. From the above experiment (Video 1), images of FRETc and bright field were merged. False positive signals were due to floating dead cells. (This may be seen in other videos)
Video 3. Smad4/dn-WWOX/p53 signaling. DU145 cells were transiently transfected with ECFP-Smad4, dn-EGFP-WWOX and DsRed-monomer-p53 expression constructs. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration). No signaling occurred.
Video 4. Smad4/WWOX/dn-p53 signaling. DU145 cells were transiently transfected with ECFP-Smad4, EGFP-WWOX and DsRed-monomer-dn-p53 [or p53(S46G)] expression constructs. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration). No signaling occurred.
Video 5. Smad4/Hyal-2(-sp)/WWOX signaling. DU145 cells were transiently transfected with ECFP-Smad4, EGFP-Hyal-2(-sp) and DsRed-monomer-WWOX expression constructs. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration). EGFP-Hyal-2(-sp) does not have a GPI sequence and is for cytosolic protein expression.
Video 6. Smad4/Hyal-2(-sp)/WWOX signaling – merged images of FRETc and bright field. From the above experiment (Video S5), images of FRETc and bright field were merged.
Video 7. Smad4/Hyal-2(as)/WWOX signaling. DU145 cells were transiently transfected with ECFP-Smad4, EGFP-Hyal-2(as) and DsRed-monomer-WWOX expression constructs. EGFP-Hyal-2(as) is an antisense construct. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration). No signaling occurred.
Video 8. Smad4/Hyal-2(-sp)/dn-WWOX signaling. DU145 cells were transiently transfected with ECFP-Smad4, EGFP-Hyal-2(-sp) and DsRed-monomer-dn-WWOX expression constructs. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration). No signaling occurred.
Video 9. ECFP/EGFP/DsRed monomer signaling. DU145 cells were transiently transfected with ECFP, EGFP and DsRed-monomer expression constructs. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration). No signaling occurred.
Video 10. IkBa/ERK/WWOX signaling. DU145 cells were transiently transfected with ECFP-IkBa, EGFP-ERK and DsRed-monomer-WWOX expression constructs. After culturing overnight, cells were treated with HA (25 mg/ml). Time-lapse FRET microscopy for interactions of 3 proteins was carried out [42]. Each picture was taken per 20 min. Data is shown as FRETc (FRET concentration). No signaling occurred.